Abstract Chemical exposure resulting in DNA damage (genotoxicity) can lead to a number of negative consequences in human populations, including germ cell mutations and cancer. As a result, evaluation of genotoxic potential is a priority requirement of regulatory agencies such as the EPA and FDA. Predictivity of the current suite of assays is limited by species differences, low sensitivity, and interference by non-relevant toxicity (cytotoxicity, apoptosis, etc.). To overcome assay limitations, a 3-test battery is used that includes bacterial, mammalian in vitro, and short-term rodent in vivo tests. Nonetheless, limitations in sensitivity and high false positive rates of current approaches often lead to equivocal results that drive the need for additional animal studies. Further, while in vitro tests are often used to classify compounds or prioritize chemicals for animal studies, the in vitro methods are not currently used quantitatively to derive points of departure for safety decisions, due to limitations in methodology and questionable relevance to in vivo response. The current gold standard for carcinogenicity is the 2-year rodent bioassay, a time- and money-intensive test that also suffers from a high false positive rate for identification of human carcinogens. A method that can rapidly and selectively identify compounds that damage DNA in the human, and addresses key limitations to the current battery, is clearly needed. We are developing a novel technique to directly label DNA double strand breaks (DSBs) with a fluorescent tag in human cells. Preliminary results indicate that this DNA damage labeling (DDL) assay has a high sensitivity, measuring responses at concentrations of positive control compounds as much as 10-fold lower than the widely accepted micronucleus assay. Because this state of the art approach directly evaluates DNA integrity and shows a high sensitivity, is not as prone to off-target effects (cytotoxicity) as the standard genotoxicity assays. Our proposed work is designed to demonstrate that the DDL assay improves identification of genotoxic compounds and provides a quantitative measure of DNA damage. Benefits of this assay over current tests are human relevance (vs. bacterial tests), improved quantitation (vs. comet), utility in non- proliferative/primary cells (vs. micronucleus) and less confounding by disruption of repair processes (p-H2AX). While it is clear that no single in vitro assay can provide a definitive method for quantitative prediction of cancer risk, this assay, which directly addresses limitations in the current battery, would provide a highly sensitive, human relevant approach that would improve weight of evidence-based quantitative safety decisions. Preliminary work focuses on a flow cytometry based measurement, but this project will also determine the feasibility of using the assay in a high content imaging platform. Such a method would have profound impacts on industry by providing a quantitative genotoxicity screen with improved resolution at low doses and improved accuracy at high doses; thereby reducing the need for animal testing and the cost of product development.